We propose to produce a subunit vaccine for Entamoeba histolytica and test it in mice for immunogenicity and for protection from intestinal amebiasis. Entamoeba histolytica is a Category B NIH Biodefense pathogen due to its low infectious dose and food and water-bone transmission, and an important cause of diarrhea in children in Africa, Asia and Latin America. The candidate vaccine antigen is the LecA domain of the E. histolytica Gal/GalNAc lectin that mediates parasite adherence and contact-dependent cytotoxicity. LecA contains all of the virulence neutralizing antibody epitopes of the native Gal/GalNAc lectin, and in fact a surrogate marker of immunity in children is intestinal IgA against LecA. Feasibility of our approach is underscored by the effectiveness of LecA in many investigators' laboratories as a vaccine in rodent models of amebiasis. Innovative aspects of the proposed research include production of the first vaccine against an enteric parasite, the use of an antigen (LecA) where virulence-neutralizing epitopes have been mapped, and utilization of the novel mouse model of amebic colitis for efficacy studies. Our approach will be to conduct preclinical development of the LecA vaccine formulated in alum. Preliminary studies published from our group have demonstrated the effectiveness of this prototype vaccine in the murine model. Two specific aims are proposed to conduct this work: In Specific Aim 1, we will express and purify from E. coli the LecA fragment of the Gal/GalNAc lectin using standard operating procedures that are scalable to cGMP. We will quality control the purified protein for endotoxin, DNA, and contaminating host cell proteins, absorb it to alum and test its stability and lot-to-lot variability. TechLab will be entirely responsible for Aim 1. In Specific Aim 2, we will compare different dosing intervals and amounts to examine the immunogenicity, both cellular and humoral, of the alum-absorbed LecA parenteral vaccine for (a) the magnitude of an antigen-specific pre-challenge IFN-g+, CD4+ T cell response to LecA, and mucosal and serum antibody responses, and (b) vaccine efficacy and durability. UVA will perform all immunizations, cell mediated immune response tests, humoral responses, and vaccine trials. TechLab will provide the LecA alum-absorbed vaccine and reagents for ELISA analysis of humoral responses. The research proposed builds on the 15 year collaboration in amebiasis of the investigators, Dr. Lyerly of TechLab and Dr. Petri (the discoverer of the Gal/GalNAc lectin) from UVa. The proximity of TechLab and UVa (a 2 hour drive on I-81) facilitates this collaborative environment. Successful completion of these studies will ready the LecA vaccine for cGMP manufacture and toxicity studies (required for future phase I human clinical trials), maximize its immunogenicity and efficacy, and thereby set the stage for phase I clinical trials of an amebiasis vaccine for humans.